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. 1998 Jun 1;26(11):2747–2753. doi: 10.1093/nar/26.11.2747

Sequence and expression characteristics of a nuclear-encoded chloroplast sigma factor from mustard (Sinapis alba).

M Kestermann 1, S Neukirchen 1, K Kloppstech 1, G Link 1
PMCID: PMC147615  PMID: 9592164

Abstract

Plant chloroplasts contain transcription factors that functionally resemble bacterial sigma factors. We have cloned the full-length cDNA from mustard (Sinapis alba) for a 53 kDa derived polypeptide that contains similarity to regions 1.2-4.2 of sigma70-type factors. The amino acid sequence at the N-terminus has characteristics of a chloroplast transit peptide. An in vitro synthesized polypeptide containing this region was shown to be imported into the chloroplast and processed. The recombinant factor lacking the N-terminal extension was expressed in Escherichia coli and purified. It confers the ability on E.coli core RNA polymerase to bind specifically to a DNA fragment that contains the chloroplast psbA promoter. Transcription of the psbA template by E.coli core enzyme in the presence of recombinant SIG1 results in enhanced formation of transcripts of the size expected for correct initiation at the in vivo start site. Together, these data suggest that the mature protein acts as one of the chloroplast transcription factors in mustard. RNA gel blot hybridization reveals a transcript at approximately 1.8 kb, which is more abundant in light-grown than in dark-grown mustard seedlings.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allison L. A., Simon L. D., Maliga P. Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants. EMBO J. 1996 Jun 3;15(11):2802–2809. [PMC free article] [PubMed] [Google Scholar]
  2. Baginsky S., Tiller K., Link G. Transcription factor phosphorylation by a protein kinase associated with chloroplast RNA polymerase from mustard (Sinapis alba). Plant Mol Biol. 1997 May;34(2):181–189. doi: 10.1023/a:1005802909902. [DOI] [PubMed] [Google Scholar]
  3. Bairoch A., Bucher P., Hofmann K. The PROSITE database, its status in 1997. Nucleic Acids Res. 1997 Jan 1;25(1):217–221. doi: 10.1093/nar/25.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brahamsha B., Haselkorn R. Identification of multiple RNA polymerase sigma factor homologs in the cyanobacterium Anabaena sp. strain PCC 7120: cloning, expression, and inactivation of the sigB and sigC genes. J Bacteriol. 1992 Nov;174(22):7273–7282. doi: 10.1128/jb.174.22.7273-7282.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brahamsha B., Haselkorn R. Isolation and characterization of the gene encoding the principal sigma factor of the vegetative cell RNA polymerase from the cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol. 1991 Apr;173(8):2442–2450. doi: 10.1128/jb.173.8.2442-2450.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burgess R. R., Travers A. A., Dunn J. J., Bautz E. K. Factor stimulating transcription by RNA polymerase. Nature. 1969 Jan 4;221(5175):43–46. doi: 10.1038/221043a0. [DOI] [PubMed] [Google Scholar]
  7. Burton Z., Burgess R. R., Lin J., Moore D., Holder S., Gross C. A. The nucleotide sequence of the cloned rpoD gene for the RNA polymerase sigma subunit from E coli K12. Nucleic Acids Res. 1981 Jun 25;9(12):2889–2903. doi: 10.1093/nar/9.12.2889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cermakian N., Ikeda T. M., Cedergren R., Gray M. W. Sequences homologous to yeast mitochondrial and bacteriophage T3 and T7 RNA polymerases are widespread throughout the eukaryotic lineage. Nucleic Acids Res. 1996 Feb 15;24(4):648–654. doi: 10.1093/nar/24.4.648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  10. Cline K., Henry R. Import and routing of nucleus-encoded chloroplast proteins. Annu Rev Cell Dev Biol. 1996;12:1–26. doi: 10.1146/annurev.cellbio.12.1.1. [DOI] [PubMed] [Google Scholar]
  11. Eisermann A., Tiller K., Link G. In vitro transcription and DNA binding characteristics of chloroplast and etioplast extracts from mustard (Sinapis alba) indicate differential usage of the psbA promoter. EMBO J. 1990 Dec;9(12):3981–3987. doi: 10.1002/j.1460-2075.1990.tb07619.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gray M. W. Origin and evolution of organelle genomes. Curr Opin Genet Dev. 1993 Dec;3(6):884–890. doi: 10.1016/0959-437x(93)90009-e. [DOI] [PubMed] [Google Scholar]
  14. Gruber T. M., Bryant D. A. Molecular systematic studies of eubacteria, using sigma70-type sigma factors of group 1 and group 2. J Bacteriol. 1997 Mar;179(5):1734–1747. doi: 10.1128/jb.179.5.1734-1747.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hedtke B., Börner T., Weihe A. Mitochondrial and chloroplast phage-type RNA polymerases in Arabidopsis. Science. 1997 Aug 8;277(5327):809–811. doi: 10.1126/science.277.5327.809. [DOI] [PubMed] [Google Scholar]
  16. Helmann J. D., Chamberlin M. J. Structure and function of bacterial sigma factors. Annu Rev Biochem. 1988;57:839–872. doi: 10.1146/annurev.bi.57.070188.004203. [DOI] [PubMed] [Google Scholar]
  17. Isono K., Shimizu M., Yoshimoto K., Niwa Y., Satoh K., Yokota A., Kobayashi H. Leaf-specifically expressed genes for polypeptides destined for chloroplasts with domains of sigma70 factors of bacterial RNA polymerases in Arabidopsis thaliana. Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14948–14953. doi: 10.1073/pnas.94.26.14948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jaehning J. A. Sigma factor relatives in eukaryotes. Science. 1991 Aug 23;253(5022):859–859. doi: 10.1126/science.1876846. [DOI] [PubMed] [Google Scholar]
  19. Jang S. H., Jaehning J. A. The yeast mitochondrial RNA polymerase specificity factor, MTF1, is similar to bacterial sigma factors. J Biol Chem. 1991 Nov 25;266(33):22671–22677. [PubMed] [Google Scholar]
  20. Kruse E., Kloppstech K. Posttranslational transport and in Thylakoido integration of plastid protein. Methods Mol Biol. 1995;37:279–291. doi: 10.1385/0-89603-288-4:279. [DOI] [PubMed] [Google Scholar]
  21. Lankiewicz S., Gisselmann G., Hatt H. Enhanced RACE method using specific enrichment by biotinylated oligonucleotides bound to streptavidin coated magnetic particles. Nucleic Acids Res. 1997 May 15;25(10):2037–2038. doi: 10.1093/nar/25.10.2037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lerbs-Mache S. The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: single-subunit enzyme or catalytic core of multimeric enzyme complexes? Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5509–5513. doi: 10.1073/pnas.90.12.5509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Link G. Cloning and mapping of the chloroplast DNA sequences for two messenger RNAs from mustard (Sinapis alba L.). Nucleic Acids Res. 1981 Aug 11;9(15):3681–3694. doi: 10.1093/nar/9.15.3681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Link G. DNA sequence requirements for the accurate transcription of a protein-coding plastid gene in a plastid in vitro system from mustard (Sinapis alba L.). EMBO J. 1984 Aug;3(8):1697–1704. doi: 10.1002/j.1460-2075.1984.tb02034.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Liu B., Troxler R. F. Molecular characterization of a positively photoregulated nuclear gene for a chloroplast RNA polymerase sigma factor in Cyanidium caldarium. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3313–3318. doi: 10.1073/pnas.93.8.3313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lonetto M. A., Brown K. L., Rudd K. E., Buttner M. J. Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmic functions. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7573–7577. doi: 10.1073/pnas.91.16.7573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lonetto M., Gribskov M., Gross C. A. The sigma 70 family: sequence conservation and evolutionary relationships. J Bacteriol. 1992 Jun;174(12):3843–3849. doi: 10.1128/jb.174.12.3843-3849.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Malhotra A., Severinova E., Darst S. A. Crystal structure of a sigma 70 subunit fragment from E. coli RNA polymerase. Cell. 1996 Oct 4;87(1):127–136. doi: 10.1016/s0092-8674(00)81329-x. [DOI] [PubMed] [Google Scholar]
  29. Masters B. S., Stohl L. L., Clayton D. A. Yeast mitochondrial RNA polymerase is homologous to those encoded by bacteriophages T3 and T7. Cell. 1987 Oct 9;51(1):89–99. doi: 10.1016/0092-8674(87)90013-4. [DOI] [PubMed] [Google Scholar]
  30. Mullet J. E. Dynamic regulation of chloroplast transcription. Plant Physiol. 1993 Oct;103(2):309–313. doi: 10.1104/pp.103.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Neuhaus H., Link G. The chloroplast tRNALys(UUU) gene from mustard (Sinapis alba) contains a class II intron potentially coding for a maturase-related polypeptide. Curr Genet. 1987;11(4):251–257. doi: 10.1007/BF00355398. [DOI] [PubMed] [Google Scholar]
  32. Newman T., de Bruijn F. J., Green P., Keegstra K., Kende H., McIntosh L., Ohlrogge J., Raikhel N., Somerville S., Thomashow M. Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol. 1994 Dec;106(4):1241–1255. doi: 10.1104/pp.106.4.1241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Oppenheimer D. G., Haas N., Silflow C. D., Snustad D. P. The beta-tubulin gene family of Arabidopsis thaliana: preferential accumulation of the beta 1 transcript in roots. Gene. 1988;63(1):87–102. doi: 10.1016/0378-1119(88)90548-3. [DOI] [PubMed] [Google Scholar]
  34. Pfannschmidt T., Link G. Separation of two classes of plastid DNA-dependent RNA polymerases that are differentially expressed in mustard (Sinapis alba L.) seedlings. Plant Mol Biol. 1994 Apr;25(1):69–81. doi: 10.1007/BF00024199. [DOI] [PubMed] [Google Scholar]
  35. Pfannschmidt T., Link G. The A and B forms of plastid DNA-dependent RNA polymerase from mustard (Sinapis alba L.) transcribe the same genes in a different developmental context. Mol Gen Genet. 1997 Dec;257(1):35–44. doi: 10.1007/s004380050621. [DOI] [PubMed] [Google Scholar]
  36. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sawadogo M., Sentenac A. RNA polymerase B (II) and general transcription factors. Annu Rev Biochem. 1990;59:711–754. doi: 10.1146/annurev.bi.59.070190.003431. [DOI] [PubMed] [Google Scholar]
  38. Shiina T., Tanaka K., Takahashi H. Sequence of hrdB, an essential gene encoding sigma-like transcription factor of Streptomyces coelicolor A3(2): homology to principal sigma factors. Gene. 1991 Oct 30;107(1):145–148. doi: 10.1016/0378-1119(91)90308-x. [DOI] [PubMed] [Google Scholar]
  39. Sugita M., Sugiura M. Regulation of gene expression in chloroplasts of higher plants. Plant Mol Biol. 1996 Oct;32(1-2):315–326. doi: 10.1007/BF00039388. [DOI] [PubMed] [Google Scholar]
  40. Tanaka K., Oikawa K., Ohta N., Kuroiwa H., Kuroiwa T., Takahashi H. Nuclear encoding of a chloroplast RNA polymerase sigma subunit in a red alga. Science. 1996 Jun 28;272(5270):1932–1935. doi: 10.1126/science.272.5270.1932. [DOI] [PubMed] [Google Scholar]
  41. Tanaka K., Shiina T., Takahashi H. Multiple principal sigma factor homologs in eubacteria: identification of the "rpoD box". Science. 1988 Nov 18;242(4881):1040–1042. doi: 10.1126/science.3194753. [DOI] [PubMed] [Google Scholar]
  42. Tanaka K., Tozawa Y., Mochizuki N., Shinozaki K., Nagatani A., Wakasa K., Takahashi H. Characterization of three cDNA species encoding plastid RNA polymerase sigma factors in Arabidopsis thaliana: evidence for the sigma factor heterogeneity in higher plant plastids. FEBS Lett. 1997 Aug 18;413(2):309–313. doi: 10.1016/s0014-5793(97)00906-x. [DOI] [PubMed] [Google Scholar]
  43. Tiller K., Eisermann A., Link G. The chloroplast transcription apparatus from mustard (Sinapis alba L.). Evidence for three different transcription factors which resemble bacterial sigma factors. Eur J Biochem. 1991 May 23;198(1):93–99. doi: 10.1111/j.1432-1033.1991.tb15990.x. [DOI] [PubMed] [Google Scholar]
  44. Tiller K., Link G. Phosphorylation and dephosphorylation affect functional characteristics of chloroplast and etioplast transcription systems from mustard (Sinapis alba L.). EMBO J. 1993 May;12(5):1745–1753. doi: 10.1002/j.1460-2075.1993.tb05822.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tiller K., Link G. Sigma-like transcription factors from mustard (Sinapis alba L.) etioplast are similar in size to, but functionally distinct from, their chloroplast counterparts. Plant Mol Biol. 1993 Feb;21(3):503–513. doi: 10.1007/BF00028807. [DOI] [PubMed] [Google Scholar]
  46. Troxler R. F., Zhang F., Hu J., Bogorad L. Evidence that sigma factors are components of chloroplast RNA polymerase. Plant Physiol. 1994 Feb;104(2):753–759. doi: 10.1104/pp.104.2.753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wang L. F., Doi R. H. Nucleotide sequence and organization of Bacillus subtilis RNA polymerase major sigma (sigma 43) operon. Nucleic Acids Res. 1986 May 27;14(10):4293–4307. doi: 10.1093/nar/14.10.4293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Weihe A., Hedtke B., Börner T. Cloning and characterization of a cDNA encoding a bacteriophage-type RNA polymerase from the higher plant Chenopodium album. Nucleic Acids Res. 1997 Jun 15;25(12):2319–2325. doi: 10.1093/nar/25.12.2319. [DOI] [PMC free article] [PubMed] [Google Scholar]

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